PROJECT SUMMARY Evolution of host defense to combat a multitude of pathogens is characterized, in part, by polarization towards either the T helper (Th) 1 or Th2 cytokine profile. The identification of theTh17 cytokine family provides a new arm of the adaptive immune response that is important for chronic inflammation. Host homeostasis relies on the delicate balance among the individual Th cell lineages that are controlled, in part, through reciprocal regulation of the cytokines. It is known that pathogen-induced up-regulation of Th1 vs Th2 cytokines induces stereotypic and sometimes opposing changes in gut smooth muscle and epithelial cell responses that are important for host resistance. An imbalance, hyper- or hypo-responsiveness, or lack of coordination among the Th cytokine profiles, is associated with dysregulation of gut function and is a characteristic of a number of mucosal inflammatory conditions of the gut including inflammatory bowel disease (IBD). Currently, there is no cure for IBD and no existing medication induces remission in all patients. One approach to control IBD is to rectify the cytokine imbalance by increasing Th2 cytokines. This may be more relevant to Crohn's disease, which is dominated by Th1/Th17 profile. IL-25 (IL-17E) is a newly-identified cytokine and a member of the IL-17 cytokine family. Unlike other cytokines in this family, IL-25 promotes Th2 while suppress Th1 and Th17, cytokine responses, thereby serving as a key regulator of inflammation in the gut mucosa. The cell types and mechanisms involved in IL-25's ability to promote Th2 and/or suppress Th1/Th17 response, particularly in the colon, are unknown. There is little information on the regulation of IL- 25 during inflammation or its contribution to the associated changes in gut function. Our preliminary data show that epithelial cells are a major source of IL-25 and also express IL-25 receptors IL-17RA and IL-17RB implicating this cytokine as a key component in mucosal immunity. The central hypothesis of this project is that epithelial derived IL-25 plays a key role in modulating mucosal immunity and barrier function as well as promoting the Th2-mediated changes in immune and gut function. There are 3 specific aims in this project. Specific Aim 1 will characterize the mechanism and outcome of nematode infection-induced up-regulation of IL-25 in colonic epithelial cells. We hypothesize that there is a reciprocal regulation between IL-4/IL-13 and IL-25 that promotes Th2-mediated host protective immunity. We will 1) identify the cell populations in the colon that produce and/or respond to IL-25 and the underlying mechanism of immune regulation of IL-25/IL- 17RA/IL-17RB in mice during T. muris infection; 2) investigate the direct induction of IL-25/IL-17RA/IL-17RB expression in colonic epithelial cell by IL-4/IL-13 activation of STAT6; 3) investigate IL-25 promoting Th2 immune response via macrophages; and 4) Determine the direct induction of IL-25 by nematode or the product in epithelial cells. Specific Aim 2 will determine the role of IL-25 in the immune regulation of colonic function. We hypothesize that IL-25 has both direct and indirect effects on epithelial and smooth muscle function that are critical to an effective Th2 protective immunity. We will 1) Determine the role of IL-25 in the physiologic and pathophysiologic control of colonic function; 2) Investigate the dependence of exogenous IL- 25-induced alterations in colonic function on IL-4, IL-13, and STAT6; 3) Establish the direct effects of IL-25 on colonic epithelial permeability; and 4) Determine the direct effects of IL-25 on colonic smooth muscle contraction. Specific Aim 3 will determine the role of IL-25 in colonic inflammation. We hypothesize that IL-25 is a critical regulator of intestinal immune homeostasis and dysregulation of IL-25 immune responses plays an important role in colonic inflammation. We will 1) Determine whether a defect in IL-25 affects the susceptibility of mice to colonic inflammation; 2) Assess the effects of exogenous IL-25 on the course of TNBS-or oxazolone-induced colitis; and 3) Investigate the role of IL-25 in modulating the innate immune response through TLR signaling. These studies provide a systematic approach to establish IL-25 as a key immuno- regulatory cytokine in the colon. In addition, these studies will establish the functional impact of IL- 25 as well as the mechanisms involved in regulation of IL-25 and IL-25 receptor expression during Th1- (TNBS-induced) and Th2- (T. muris-induced) dominant pathologies.